Chlamydia trachomatis causes ocular and genital infections in humans, which can lead to blindness and infertility. Chlamydia pneumoniae infections are a cause of pneumonia and a risk factor for chronic obstructive pulmonary disease, asthma, and atherosclerosis. Despite their immense impact on human health, relatively little is known about the genetics of Chlamydia pathogenesis, because no genetic techniques exist to create targeted mutants in these organisms. Thus there is a pressing need for the development of genetic systems to manipulate the chromosome of Chlamydia. We have created plasmids based on pCHL1, the natural plasmid found in C. trachomatis, and this will provide the basis of a gene disruption system to initiate genetic manipulation of this elusive group of bacteria. We will use this plasmid as the basis for the development of genetic manipulation techniques in Chlamydiae. We will first optimize transformation conditions to isolate both C. trachomatis and C. muridarum carrying the plasmid. We will then adapt the plasmid to deliver a Group II intron into Chlamydia, to facilitate site-specific gene inactivation. Successful transformation and genetic modification of Chlamydia species will propel the study of this important group of human pathogens forward, and allow for the development of novel therapeutics and vaccines against these bacteria.